Download reduced expression of tissue transglutaminase in a human

550s Biochemical SocietyTransactions ( 1996) 24
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THE ACTIVITY OF INTERFERON-? IS DEPENDENT ON
SEQUESTRATION BY CELL-SURFACE HEPARAN SULPHATE
m.M.S., Rix, D.A., Talbot, D. and Kirby, I.A.
Transplant Immunology Unit, Department of Surgery, The Medical School,
University of Newcastle, Newcastle upon Tyne NE2 4HH.
EFFECT OF SHORT- AND LONG-TERM ADRENERGIC
STIMULATION ON THE EXPRESSION OF THE UNCOUPLING
PROTEIN THERMOGENIN (UCP) IN BROWN ADIPOCYTE
PRECURSOR CELLS DIFFERENTIATING IN CULTURE
DavidHerron,
Sklton Mariga, Pen Puigserver, Barbara Cannon
and Jan Nedergaard.
The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm
University. S-106 91 Stockholm, Sweden
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Interferon-y (IFN-y) is an important cytoldne involved in regulation of the
activation, growth and differentiation of immune cells and other cell types such
as those of the endothelium. Previously we have demonstrated that IFN-y
activity is antagonised by heparin, a heavily dphated glyco&noglycan
(GAG), and the structurally related molecule heparan dphate. More recently,
using a oon-anticoagulant heparin derivative (Leo Pharmaceuticals), we have
shown that the ability of heparin to inhibit IFN-y is unrelated to its
anticoagulant activity. In this present study, we have investigated the
underlying mechanism for IFN-y inhibition by heparin.
Radioligand binding assays demonstrated that total binding of
'z'I-labelled IFN-y to the EAhy.926 endothelial hybridoma cell line was
significantly reduced in the presence of heparin or heparan sulphate at
125pglml; the structurally dissimilar GAG chondroitin sulphate had no effect.
This suggests that soluble heparin inhibits IFN--, by competing with heparinlike, cell-surface GAGs to bind the cytoldw.
Treatment of EAhy.926 cells with 25mM chlorate was non-toxic but
inhibited the incorporation of ["S]-sulphate into GAG chains, and greatly
reduced the capacity of these cells to bind [lUI]-IFN-y; this was paralleled by
decreased upregulation of class I1 MHC antigens by IFN-y stimulated cells that
had been pre-treated with chlorate. This indicates that sulphated, negatively
charged regions on cell-surface GAGs are involved in both the sequestration
and biological activity of IFN-y.
A cationic 10 amino acid peptide sequence (-AKTGKRKRSG-) from
the C-terminal region of IFN-y was found to competitively reduce binding of
['"I]-IFN-y to the cell line. This provides evidence for interaction between a
specific region of the cytokine molecule and cell-surface GAGs.
These results indicate that IFN-y is sequestered onto endothelial cells
by binding to sulphated, heparin-like domains on cell-surface heparan sulphate
proteoglycans. This interaction appears to be essential for optimal cytokine
activity and represents a potential target for clinical immune modulation.
Brown adipose tissue (BAT) is a thennogenic organ present in most mammals.
During cold acclimation, BAT recruits (a process involving both the
proliferation and differentiation of brown adipocyte precursor cells) and
becomes functionally more active. In-vivo and recent in-vitro experiments
stongly suggest a central role for the neurotransmittor noradrenaline in the
regulation of this cold-induced BAT recruitment. We are currently investigating
the effects of noradrenaline on mouse brown adipocyte precursor cells
differentiating in culture in order to further investigate this hypothesis.
Differentiating mouse brown adipocyte precursor cells were treated from
in the
I)
short-term (24h; acute) or in
culturing day 7 with noradrenaline (10 @
the longer-term (daily additions for up to 5 days; chronic) and the expression of
UCP determined by immunoblotting on culturing days 8 - 12. Acute adrenergic
stimulation led to maximal UCP expression on day 8 and thereafter
noradrenaline-induced UCP expression declined suggesting a thermogenic
dedifferentiation of the cells with time in culture. In clear contrast, chronic
adrenergic stimulation led to ever increasing UCP expression up to culturing
day 12. Thus, chronic adrenergic stimulation not only prevents but also reverses
the apparent thermogenic dedifferentiation which occurs in mouse brown
adipocyte cultures.
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REDUCED EXPRESSION OF TISSUE
TRANSGLUTAMINME IN A E"ENDOTHELIAL
CELL LINE LEADS TO CHANGES IN CELL
SPREADING AND CELL ADHESION
EVIDENCE FOR A ROLE FOR
SENESCENCE
AND
P16 IN REPLICATIVE
IMMORTALI'IY
IN
HUMAN
KERATINOCYTES.
0,Malliri A, Owens D, Ozanne B. Frame M, Parkinson
EK. CRC Beatson Laboratories, Garscube Estate, Switchback Road,
Jones. R. A,, Nicholas, B., Man, S m d Griffin, M.
Bearsden,Glasgow G61 1BD.
Department of Life Sciences, Nottingham Trent University,
Clifton Lane, Clifton, Nottingham, NGl 1 8NS, U. K
Two candidate tumour supressor genes have been mapped to 9 2 1 . a
region conundy lost in tumours. These candidate genes are p16 and
p15. W e have studied a series of squamous cell carcinomas of the
head and neck (SCC-HN) with respect to p15 and p16. W e have
found that LOH on 9p21 is a common event in SCC. W e have also
found that all of our immortal cell lines have undetectable expression
of p16. This appears to be due to either homoygous deletion of the
gene o r transcriptional silencing of the gene by methylation. W e have
observed no mutations in the coding sequence of any of the lines
retaining p16. Reintroduction of p16 into immortal cell lines causes a
reduction in cloning efficiency. In contrast the p15 gene was rarely
transcriptionally silenced and was not homoygously deleted in any of
the cell lines showing deletion of p16. The status of pRb appears
normal in all our cell lies. W e have also found that in contrast to
immortal cells, normal human kern- acaunulate p16 to large
amounts as they approach replicative senescence. The. status of the
d l s has been c o n f i i using (3-galactosidaee staining. However
there appears to be no increase in p16 in cells undergoing terminal
differentiation induced by treatment with calcium and monitored by
staining for involuain. These results suggest that p16 is an important
elanent of cell cycle control being implicated in replicative
and the loss of p16 appearing to be important for progression towad9
immorlalilyin SCC-HN.
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Tissue transglutaminase (tTGase) is a Ca2'dependent GTPbinding enzyme Which CrOSSlinkS proteins Via E(y-glUtaUlyl)
bridges. The physiological functions of tTGase have yet to be
elucidated, but recent evidence suggests that the enzyme may
play a rok m extracehhr matrix organisation where it
crosslinks matrix proteins such as fibronectm, collagen, laminin
and osteonectin.
In order to study the enzymes function m the endothelial cell line
ECV304, tTGase expression was reduced by stable transfection
with a 1.1 kb antisense construct m the plasmid vector pSG5.
ECV304 clones carrying the antisense construct showed
reductions m tTGase activity of 75 and 90%. These clones
exhiied phenotypic differences compared to the parent line and
transfected controls which included reduced cell spreading and
an mcreased susceptibilityto detachment by trypsm.
Reduced cell spreading was accompanied by a decrease m the
production of fibronectm polymer when analysed by
imrnunoprobing of western blots, which could be correlated to
the amount of tTGase externalised by these cells when measured
by biotmyiated cadaverine mcorporation mto fibronectin.